| Objective:Determining the affection of Mucin 13(MUC13)on the biological behavior such as proliferation,cell cycles,apoptosis,migration,and invasion of cells of human laryngeal squamous cell carcinoma.Obtain expression conditions and clinical significance of MUC13in laryngeal squamous cell carcinoma.Methods:1.MUC13-RNAi(RNA interference)was obtained by siRNA sequences screening or MUC13 in human laryngeal squamous cell carcinomacell lines.In order to specifically decrease the expression of MUC13 the above selected siRNA sequence was packaged into lentivirus and transfected into human laryngeal squamous cell carcinoma cell line Tu177.Q-PCR and western blotting were applied to determine the inhibition efficiency on MUC13 expression ofMUC13-RNAilentivirus transfected Tu177 cells.CCK-8 assay,tablet cloning assay,transwell assay,and flow cytometry were applied to confine the affection of MUC13 expression inhibition on the proliferation,cycle,migration,invasion behaviors of Tu177 cells.2.From June 2016 to April 2020,93 cases of laryngeal cancer tissue samples,49 cases of paracancerous tissue samples,19 cases of intraepithelial neoplasia tissue samples and 39 cases of vocal cord polyp tissue samples were collected from patients with laryngeal cancer or other diseases who underwent Otolaryngology Head and Neck Surgery in North Sichuan Medical College.Immunohistochemical staining was used to detect the expression of MUC13 protein in the above four kinds of tissues.The clinical data of the above patients were collected during the same period to determine the relationship between the expression of MUC13 in laryngeal cancer tissues and the clinicopathological factors of the patients.At last,the prognostic factors of the patients were analyzed.3.All the above immunohistochemical data were analyzed using IBM SPSS Statistics 25.And P<0.05 indicates that the difference is statistically significant.Results:1.After been confirmed by q-PCR,and western blotting determinations,the expression of MUC13 in Tu177 was significantly reduced by transfecting.The proliferation,cycle,migration,invasion behaviors of Tu177 cells were all affected by inhibiting the expression of MUC13.Compared with the Blank control(Blank)Tu177 cells and the meaningless sequence transfected negative control(NC)Tu177 cells,the OD value variation rate of CCK-8 test of Tu177 cells in the MUC13-RNAi group decreased with culture time.The relative clone formation rate of Tu177 cells in the NC group was 78.9%compared with that in the blank group and that of Tu 177 cells in the MUC13-RNAi group was 54.1%.And then the relative clone formation rate of Tu177 cells in the MUC13-RNAi group was significantly decreased(P<0.05).In the migration experiment,the relative migration rate of laryngeal cancer cells in the NC group was 94.5%when compared with that of the blank group,and the difference was not statistically significant(P>0.05).Compared with the blank group,the relative migration rate of Tu177 cells in the MUC13-RNAi group was 53.2%,and the relative migration rate of Tu177 cells in the MUC13-RNAi group was 56.3%when compared with that of NC group,with statistical significance(P<0.05).The migration ability of Tu177 cells in the MUC13-RNAi group was significantly decreased.In the invasion experiment,compared with blank group,the relative migration rate of laryngeal cancer cells in NC group was 95.2%,the difference was not statistically significant(P>0.05).Compared with the blank group,the relative migration rate of laryngeal cancer cells in the MUC13-RNAi group was 65.9%,and the relative migration rate of laryngeal cancer cells in the MUC13-RNAi group was 69.2%when compared with that of NC group,the differences were statistically significant(P<0.05).The invasion ability of Tu177 cells in the MUC13-RNAi group was significantly decreased.At the same time,Tu177 cells in the MUC13-RNAi group experienced G1 phase arrest and increased tendency of early apoptosis,indicating that the proliferation ability of Tu177 cells in the MUC13-RNAi group was decreased,which was consistent with the results of CCK-8 and cell clone formation experiments.2.The results of MUC13 Immunohistochemical staining showed that the average IOD value of laryngeal cancer tissue was 0.0445±0.0254,the average IOD value of laryngeal intraepithelial neoplasia tissue was 0.0214±0.0155,the average IOD value of paracancerous tissue was 0.0095 ± 0.0107,and the average IOD value of vocal cord polyp tissue was 0.0069± 0.0085.It could be concluded that the expression of MUC13 increases in the process from normal laryngeal squamous epithelium and early laryngeal lesions to laryngeal squamous neoplasia and then to laryngeal squamous cell carcinoma.There was no significant correlation between the expression level of MUC13 in laryngeal cancer tissues and gender,age,tumor site and lymph node metastasis of the patients(P>0.05).Expression level of MUC13 was significantly related to T stage,clinical stage and degree of differentiation,MUC13 expression in tumor ofhigh differentiation(IOD0.0513 ± 0.0242),T3～4(IOD 0.0539±0.0268)and clinical staging Ⅲ-Ⅳ(IOD0.0518±0.02728)than those in tumor oflow differentiation(IOD 0.0372±0.0246),T1～2(IOD 0.0395 ± 0.0232)and clinical stage Ⅰ～Ⅲ(IOD 0.0401±0.0232).The differences were statistically significant(P<0.05).Kaplan-Meier survival curve indicated that the survival time of patients with MUC 13 expression greater than or equal to the mean was significantly lower than that ofpatients with MUC 13 expression less than the mean,and the difference was statistically significant(P<0.05).Sub and lower glottis type,higher clinical stage,higher T grade,lymph node metastasis and high MUC13 expression were the risk factors affecting the survival time of patients with laryngeal cancer.However,in Cox regression analysis,the significance P values of the above 5 factors were all greater than 0.05,so they could not be determined as independent risk factors affecting the survival time of patients with laryngeal cancer.Conclusions:1.After the specific inhibition of MUC13 expression in the laryngeal squamous cell carcinoma line Tu177,the proliferation ability,proliferation and migration ability of the cells were decreased,the cells were slightly G1 blocked and the apoptotic tendency was increased,suggesting that MUC 13 is involved in the regulation of a variety of biological behaviors of laryngeal squamous cell carcinoma cells.2.The expression level of MUC 13 in vocal cord polyps and paracancerous tissues,laryngeal squamous epithelial neoplasia tissues and carcinoma tissues increased successively,in another word increased with the increase of clinical stages.The expression level of MUC 13 in highly differentiated laryngeal squamous cell carcinoma tissues was higher than that in medium-low differentiated tissues,indicating that MUC13 is related to the occurrence and development of laryngeal squamous cell carcinoma.MUC13 has certain guiding significance in predicting the prognosis of laryngeal squamous cell carcinoma,but the mechanism of its role in laryngeal squamous cell carcinoma still needs further study. |
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